CN116836848A - 一种能够改善功能性便秘的嗜酸乳杆菌及应用 - Google Patents
一种能够改善功能性便秘的嗜酸乳杆菌及应用 Download PDFInfo
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Abstract
本发明提供了一种能够改善功能性便秘的嗜酸乳杆菌及应用，所述嗜酸乳杆菌经中国微生物菌种保藏管理委员会普通微生物中心保藏，保藏日期为2023年2月20日，保藏编号为CGMCCNO.26581。本发明所述嗜酸乳杆菌ZJPY001对酸、胆盐具有良好的耐受性，对大肠杆菌、金黄色葡萄球菌的生长均有明显的抑制作用，而对双歧杆菌的生长有一定的促进作用，同时动物实验表明其对功能性便秘具有一定的治疗和预防效果。
Description
技术领域
发明涉及微生物技术领域，具体而言，涉及一种能够改善功能性便秘的嗜酸乳杆菌及应用。
背景技术
功能性便秘(functional constipation，FC)，是指非全身性疾病或肠道疾病所引起的，无器质性损伤，无组织结构异常或代谢障碍，又除外肠易激综合征的原发性持续性便秘，是功能性胃肠病最常见的类型之一，也是一类可严重影响人类生活质量的常见疾病。主要临床表现为排便次数减少、持续性排便困难、粪便干结、排便费力或排便不尽感等，同时患者通常存在有肠动力功能及排便生理功能异常。虽然临床表现的个体化差异较大，但一般认为与肠道菌群失调密切相关。
随着益生菌与人体健康的研究日渐受到全球关注，通过补充益生菌调节肠道菌群，最终实现便秘的缓解则逐渐成为治疗便秘的重要手段。嗜酸乳杆菌(Lactobacillusacidophilus)是自然存在于人体体内的益生性细菌，主要位于人体肠胃道中，其能够维持体内菌相平衡的环境，预防以及抑制有害细菌生长，避免有害细菌危害人体健康。各种功能不同的嗜酸乳杆菌的新菌株不断被发现，例如降解生物胺、抑制幽门螺旋杆菌、抗衰老、抗过敏等方面，在预防和治疗疾病方面发挥重要作用；寻找耐酸耐胆盐的乳酸新菌种对于解决功能性便秘至关重要。
有鉴于此，特提出本发明。
发明内容
为解决上述问题，本发明提供一种能够改善功能性便秘的嗜酸乳杆菌(Lactobacillus acidophilus)ZJPY001，所述嗜酸乳杆菌的保藏编号为CGMCC NO.26581。
上述嗜酸乳杆菌在制备改善功能性便秘的生物制剂中的应用。
本发明还提供了一种微生物制剂，所述微生物制剂包括上述的嗜酸乳杆菌。
优选的，所述微生物制剂为菌粉，其含量不低于1.2*1012cfu/g。
优选的，所述微生物制剂的制备方法为：步骤1)种子一级培养：将筛选出的嗜酸乳杆菌ZJPY001菌种先接入灭菌后三角瓶液体培养基中，装液量为0.65-0.75，接种后在35℃下静止培养16h，即种子液；
步骤2)、种子二级培养：按照4-6％接种比加入步骤1)得到的种子液，在36-40℃下搅拌培养16-20h，搅拌转速90-110rpm，通过部件泵流加2mol/L的NaOH来控制为7.3，通入氮气维持种子罐压力在0.05MPa。
步骤3)、发酵罐培养：将二级种子罐培养得到的所述一级种子液全部泵入发酵罐，35-40℃下搅拌培养15-20h，搅拌转速180-240rpm，pH采用2mol/L的NaOH通过种子罐补碱泵自动控制在4.4-4.6，且通入氮气维持发酵罐压力在0.05MPa；培养期间通过补料泵确保罐内葡萄糖浓度在2g/L以上；
步骤4)、菌体分离、复配：通过碟片式离心机分离菌体，之后用无菌水洗涤2次得到菌泥，按照质量比5-10:1的比例将菌泥与菌泥保护剂混合乳化，进行冷冻干燥。
优选的，所述微生物制剂的制备方法为：
步骤1)种子一级培养：将筛选出的嗜酸乳杆菌ZJPY001菌种先接入1L三角瓶液体培养基，装液量为0.65，即培养基体积/三角瓶容积＝0.65，121℃灭菌20min，接种后在35℃下静止培养16h，即种子液；步骤2)、种子二级培养：按照4％接种比加入步骤S1得到的种子液，在36℃下搅拌培养16h，搅拌转速110rpm，通过部件泵流加2mol/L的NaOH来控制为7.3，通入氮气维持种子罐压力在0.05MPa。
步骤3)、发酵罐培养：将二级种子罐培养得到的所述一级种子液全部泵入发酵罐，35℃下搅拌培养18h，搅拌转速200rpm，pH采用2mol/L的NaOH通过种子罐补碱泵自动控制在4.4-4.6，且通入氮气维持发酵罐压力在0.05MPa；培养期间通过补料泵确保罐内葡萄糖浓度在2g/L；
步骤4)、菌体分离、复配：以8000rpm条件下进行以碟片式离心机分离菌体，并以无菌水洗涤2次，得到菌泥。按照质量比6:1的比例将菌泥与菌泥保护剂混合乳化，进行冷冻干燥。
相对于现有技术，本发明所述嗜酸乳杆菌具有如下有益效果：1)本发明提出的保藏编号为CGMCC No.26581的嗜酸乳杆菌ZJPY001对酸、胆盐耐受性好，同时能够改善肠道菌群并对功能性便秘具有一定的治疗和预防效果；2)通过三级发酵并控制pH、葡萄糖控制可实现高密度发酵，制备菌粉中菌含量可达1012CFU/g。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例所述菌株ZJPY001的菌落形态图；
图2为本发明实施例所述菌株ZJPY001对酸的耐受性结果；
图3为本发明实施例所述菌株ZJPY001对胆盐的耐受性结果。
具体实施方式
为使本发明的上述目的、特征和优点能够更为明显易懂，下面结合附图对本发明的具体实施例做详细的说明。需要说明的是，本发明实施例中的技术特征可以相互组合。
功能性便秘是最常见的肠道疾病之一。长期便秘可引发肛周疾病、结肠黑便、结肠憩室及泻剂结肠等，且便秘可诱发多种心脑血管疾病。严重者还会伴有烦躁、失眠、抑郁、强迫观念和行为、易怒等精神行为异常。临床上通常使用药物或手术进来治疗便秘，但药物治疗容易引起抗药性和其他并发症，而手术治疗通常治标不治本而易反复。
实施例1嗜酸乳杆菌的分离筛选及鉴定
1、分离筛选
菌株分离自健康人体粪便，具体分离筛选方法如下：取0.5g粪便样品至10mL离心管中，加入6mL生理盐水后充分震荡分散；取100μl分散液加入生理盐水依次稀释至10-2、10-3、10-4、10-5，各取100μl涂布于MRS固体培养基上，37℃培养48-72h后挑取典型菌落，用接种环挑取2-3环转接到pH值为6.5-7.0的液体MRS培养基中，37℃需氧培养24h后，按4％的比例分别接种于pH＝3.0的酸性的MRS液体培养基中进行需厌氧培养，初步筛选获得耐酸特性菌株，对其镜检观察并进行初步筛选和纯化培养，得到ZJPY001的菌株。
2、形态学鉴定
菌株ZJPY001在MRS固体培养基中培养24h后，菌落呈白色，圆形，不透明，边缘整齐，结果如图1；在MRS液体培养基培养6h后明显浑浊，久置后液体培养基底部出现呈白色沉淀，革兰氏染色为阳性。
3、16SrDNA基因序列分析：
挑取单菌落置MRS液体培养基中，37℃培养过夜后，12000转离心1min收集菌体，按照DNA提取试剂盒步骤进行操作。引物采用细菌通用引物27F，1492R，PCR扩增体系为50μL体系，95℃预变性5min；94℃15s，57℃15s，72℃40s，35个循环；72℃延伸10min。
PCR产物测序结果与GenBank中已发表的标准序列进行同源性比较(BLASTN)后得出ZJPY001菌株为嗜酸乳杆菌(Lactobacillus acidophilus)，于2023年2月2日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101，保藏编号为CGMCC No.26581，菌株编号为ZJPY001。
实施例2耐酸耐胆盐性能
2.1耐酸检测
将含1012CFU/ml嗜酸乳杆菌ZJPY001的菌粉分别接种到pH值为2.0、3.0、4.0、5.0的MRS培养基中，分别在2h、4h、6h、8h采用平板倾注法测定其活菌数。
当处于酸性环境即pH为5.0、4.0、3.0时，嗜酸乳杆菌可以正常生长，当pH为2.0时，对嗜酸乳杆菌的生长略有抑制作用，但活菌数仍然能维持在10.6lg(cfu/ml)，说明嗜酸乳杆菌ZJPY001可以耐受胃酸的影响，如图2所示。
2.2、耐胆盐检测
将活化好的嗜酸乳杆菌ZJPY001用无菌生理盐水做倍比稀释，选取合适的稀释梯度并吸取200μl稀释液放于无菌培养皿里，做4个重复，然后用含不同浓度牛胆酸钠(0.1％、0.2％、0.3％、0.4％)的MRS培养基倾注平板，37℃培养8小时，每隔2小时菌落计数，同时用不含牛胆酸钠的MRS培养基倾注平板，37℃培养48小时，菌落计数，作为对照组。
结果显示，0.1-0.3％的胆盐作用对嗜酸乳杆菌ZJPY001的影响比较微弱，几乎不影响其正常生长。在0.4％的胆盐作用8小时后，活菌数略有下降，说明嗜酸乳杆菌ZJPY001具有较强的耐胆盐能力，如图3所示。
实施例3菌粉的制备
步骤1)、种子一级培养
将筛选出的嗜酸乳杆菌ZJPY001菌种先接入1L三角瓶液体培养基，装液量为0.65，即培养基体积/三角瓶容积＝0.65，121℃灭菌20min，接种后在35℃下静止培养16h，即种子液。
所述三角瓶液体培养基的组成为：蛋白胨1.0％、牛肉粉1.0％、酵母粉0.5％、葡萄糖2.0％、硫酸镁0.01％、醋酸钠0.5％、柠檬酸铵0.2％、磷酸氢二钾0.2％、硫酸锰0.005％、吐温80 0.1％，调pH值为6.0。
步骤2)、种子二级培养
二级种子罐体积为20L，装液量为0.7，121℃灭菌20min，按照4％接种比加入步骤S1得到的种子液，在36℃下搅拌培养16h，搅拌转速110rpm，通过部件泵流加2mol/L的NaOH来控制为7.3，通入氮气维持种子罐压力在0.05MPa。
所述二级种子罐的培养基组成为：酵母浸粉1.0％、胰蛋白肽0.5％、大豆蛋白肽1.0％、牛肉浸粉0.5％、果糖0.4％、维生素C 0.05％、无水醋酸钠0.05％、磷酸氢二钾0.04％、柠檬酸二铵0.02％、七水硫酸镁0.006％、四水硫酸锰0.003％、吐温80 0.01％,调pH值为6.5。
步骤3)、发酵罐培养
发酵罐体积为500L，装液量为0.6，121℃下30min；将二级种子罐培养得到的所述一级种子液全部泵入发酵罐，35℃下搅拌培养18h，搅拌转速200rpm，pH采用2mol/L的NaOH通过种子罐补碱泵自动控制在4.4-4.6，且通入氮气维持发酵罐压力在0.05MPa，连续检测三次OD600值不变化确切大于为2.0，镜检耐酸乳杆菌形态正常，无杂菌，终止发酵。
培养期间每隔30min取样检查葡萄糖残余浓度，通过补料泵补充葡萄糖以维持罐内葡萄糖浓度在2g/L；所述发酵罐培养基的配方为：酵母浸粉1.0％、胰蛋白肽0.5％、大豆蛋白肽1.0％、牛肉浸粉0.5％、果糖0.4％、维生素C 0.05％、无水醋酸钠0.05％、磷酸氢二钾0.04％、柠檬酸二铵0.02％、七水硫酸镁0.006％、四水硫酸锰0.003％、吐温80 0.01％,调pH值为6.5。
步骤4)、菌体分离、复配
以8000rpm条件下进行以碟片式离心机分离菌体，并以无菌水洗涤2次，得到菌泥。按照质量比6:1的比例将菌泥与菌泥保护剂混合乳化，进行冷冻干燥，用双铝膜密封真空包装，每袋100g，产品中活菌数＞1.0×1012cfu/g。所述菌泥保护剂的配方为：脱脂奶粉10％、海藻糖7％、甘油5％、抗坏血酸钠0.1％、氯化钠0.3％、明胶0.1％，115℃条件下灭菌30min后冷却至5～20℃备用。
优选的，所述冷冻干燥采用分段冷冻干燥方法进行，包括：S1、将所述乳化液在-40℃的温度条件下冷冻100min；S2、之后在-20℃和30Pa下冷冻60min；S3、再在0℃和1000Pa的温度和压力条件下冷冻80min；S4、将混合物以0.3℃/min的升温速率和60Pa/min的升压速率将温度和压力升高至10℃和10kPa的条件下，保持420min，得到高活性嗜酸乳杆菌冻干菌粉。通过分段冷冻干燥，能够使得嗜酸乳杆菌菌体得以完整的保留，保证其具有良好的生物活性。
实施例4抑菌实验
分别将公司实验室保存的大肠杆菌、金黄色葡萄球菌，双歧杆菌接种于营养肉汤，37℃摇床培养过夜；每5mL灭菌营养琼脂培养基(50℃左右)加100μL致病菌菌液(106cfu/ml)，混匀后倾倒至营养琼脂平板做成双层平板，凝固后在培养基上放置牛津杯，将实验分成四组，分别向牛津杯中添加200μL由本申请中实施例3制备菌粉稀释得到的菌液(107cfu/ml)、以及申请号201910188089.1中实施例1方法制备的菌剂稀释得到的菌液(107cfu/ml)、青霉素及生理盐水，待菌液扩散后放入37℃培养箱中培养20h，观察抑菌圈直径，结果见表1。
3实验结果
表1不同样品的抑菌效果
由上表可知，嗜酸乳杆菌ZJPY001对大肠杆菌、金黄色葡萄球菌均有明显的抑制作用；但对双歧杆菌这一益生菌无抑制作用，甚至对其生长有一定的促进作用。
实验例2：对肠道传输、动力功能的调节
1实验材料
1.1主要试剂
复方地芬诺酯，为止泻药，每片含硫酸阿托品0.025mg，盐酸地芬诺酯2.5mg(购自成都第一制药有限公司)；
100目活性炭粉，购自成都瑞丽神火木炭有限公司；
羧甲基纤维素钠，购自华唯纤维素有限公司；
1.2溶液配制
复方地芬诺酯混悬液：将30℃蒸馏水与研磨成细粉的复方地芬诺酯片以1:1的比例混匀制成复方地芬诺酯混悬液，灌胃前均需充分摇匀；
墨汁：取烧杯加入800ml纯化水，再加入羧甲基纤维素钠50g，搅拌后文火煮沸至溶液透明；再加入50g 100目的活性炭粉，搅拌均匀，文火煮沸3次(即煮沸后即刻关火，待溶液冷却至室温后再次煮沸，如此反复操作三次)后，待溶液冷却至室温后加自来水搅匀定容至1000ml，留存于0℃～5℃的冰柜，使用前充分摇匀。
2实验方法
2.1制作功能性便秘模型
选用成年的普通级健康家兔进行适应性喂养7天，采用复方地芬诺酯混悬液经口灌胃的方法制作功能性便秘模型。具体方法为：按7.5mg/kg/d的剂量家兔进行复方地芬诺酯混悬液经口灌胃，选择5只家兔作为空白组，给予相同剂量的蒸馏水灌胃。各组家兔均1次/d，连续灌胃10d。期间各组家兔每笼2只饲养，自由进食饮水，保持饲养环境良好通风，温度保持在23-25℃，相对湿度在50％-70％，12h明暗交替自然光暗周期。每日按时清理粪池槽、清洗饮水瓶及食盒，保持食物饮水干净新鲜，并密切观察家兔进食、排便情况及体重变化。
造模第10天灌胃后家兔禁食不禁水，24h后予以活性炭溶液灌胃，剂量为10ml/kg，随后恢复正常饮食饮水。从活性炭溶液灌胃完毕开始计时，记录每只家兔的首粒黑便时间、家兔24h大便粒数及24h大便质量，各组与空白对照组比较数据差异显著，即建模成功。
2.2分组、给药治疗
采用随机数字表法对采用复方地芬诺酯混悬液经口灌胃的15只家兔进行分组，分为实验1组、对照组、阳性对照组，共3组，每组5只。从11天开始，继续对各组家兔按3.5mg/kg/d的剂量进行复方地芬诺酯混悬液灌胃。同时每天对实验1组、对照组分别灌胃实施例3制备的菌粉及申请号201910188089.1中公开菌株制备的菌剂，阳性对照组则用等剂量的蒸馏水代替菌粉灌胃处理；另取5只家兔作为空白组，仅用等剂量的蒸馏水灌胃处理。造模第15天灌胃后家兔禁食不禁水，24h后予以活性炭溶液灌胃，剂量为10ml/kg，随后恢复正常饮食饮水。
2.3观察指标及测定方法
2.3.1家兔肠道传输功能
造模第15天后实验1组、对照组家兔禁食不禁水24h后，采用活性炭溶液灌胃，灌胃容量为10ml/kg，随后恢复正常进食进水。从墨汁灌胃的时间开始记录每只家兔的首粒黑便时间以及24h大便粒数及大便质量，结果见表2。
2.3.2胃肠激素检测
胃泌素的测定：股动脉采血2ml，常温静置15分钟，采用KDC-40低速离心机进行离心(10min，3000r/min)，吸弃上清液-30℃中保存待测；
胃动素的测定：取家兔股动脉血4ml，置于含30ul 10％EDTA二钠和30ul抑肽酶的抗凝管中颠倒混匀，采用KDC-40低速离心机进行离心(4℃，20min，3000r/min)，分离血浆，并贮存于-30℃冰箱内，为避免血浆中冷凝蛋白对胃动素测定的干扰，测定前将血浆放置于室温或冷水中复融并混匀，再次离心(4℃，5min，3000r/min)，取上清液测定。血清胃泌素、血浆胃动素的测定均采用ELISA法检测，具体步骤按照试剂盒说明书进行。
3.实验结果
3.1对功能性便秘家兔肠道传输功能的影响
各组家兔经适应性喂养后，精神状态良好，反应灵活，皮毛光泽，大便颗粒大小适中，水润有光泽；体重约为2.5～3.3kg/只，进食量约为160～190g/只/天。造模10天后，造模组家兔逐渐出现活动减少，反应迟钝，进食量减少，被毛部分脱落，大便颗粒形小、干硬等表现。而空白组家兔精神状态良好，反应灵活，皮毛光泽，大便水润有光泽。各组家兔的健康状况均良好。
表2不同样品对功能性便秘家兔肠道传输功能的治疗数据
由表2所示，与空白组家兔相比，当给家兔灌喂10天的复方地芬诺酯混悬液后，家兔的首粒黑便时间由198.4min增加至206.0-221.3min；同时经灌喂复方地芬诺酯混悬液家兔的24h大便粒数及24h大便质量均明显减少，说明通过对家兔灌喂10天的复方地芬诺酯混悬液可建立功能性便秘模型。
通过口服实施例3中制备的嗜酸乳杆菌菌粉，家兔的功能性便秘情况极大改善，首粒黑便时间由289.8min缩短至219.6min，与空白组家兔的首粒黑便时间基本一致。同时，24h大便粒数及24h大便质量均较服用前明显提高，与空白组接近，且由于对照组。
3.2对功能性便秘家兔胃肠激素的影响
胃泌素与胃动素均能促进胃肠平滑肌收缩，促进胃肠动力，二者分泌紊乱可能导致胃肠动力障碍，从而与功能性便秘亦是关系密切。
表3不同样品对功能性便秘家兔的胃动素、胃泌素分泌的影响
| 组别 | 数量(只) | 胃动素(ng/L) | 胃泌素(ng/L) |
| 空白组 | 10 | 71.2±7.1 | 141.9±17.2 |
| 实验组 | 10 | 73.1±6.9 | 132.5±25.9 |
| 对照组 | 10 | 61.9±4.3 | 110.7±15.4 |
| 阳性对照组 | 10 | 54.4±6.8 | 77.2±10.1 |
由表3可知，实验组中灌胃本发明制备的嗜酸乳杆菌均分后家兔的胃动素、胃泌素含量与空白组相当，说明经过服用一段时间后家兔的胃肠动力基本恢复。
虽然本发明披露如上，但本发明并非限定于此。任何本领域技术人员，在不脱离本发明的精神和范围内，均可作各种更动与修改，因此本发明的保护范围应当以权利要求所限定的范围为准。
Claims (5)
1.一种能够改善功能性便秘的嗜酸乳杆菌(Lactobacillusacidophilus)ZJPY001，其特征在于，所述嗜酸乳杆菌的保藏编号为CGMCCNO.26581。
2.权利要求1所述的嗜酸乳杆菌在制备改善功能性便秘的生物制剂中的应用。
3.一种微生物制剂，其特征在于，所述微生物制剂包括权利要求1所述能够改善功能性便秘的嗜酸乳杆菌。
4.根据权利要求3所述的微生物制剂，其特征在于，所述微生物制剂为菌粉，其中所述能够改善功能性便秘的嗜酸乳杆菌的含量不低于1.2*1012cfu/g。
5.根据权利要求4所述的微生物制剂，其特征在于，所述微生物制剂的制备方法为：
步骤1)种子一级培养：将筛选出的嗜酸乳杆菌ZJPY001菌种先接入灭菌后三角瓶液体培养基中，装液量为0.65-0.75，接种后在35℃下静止培养16h，即种子液；
步骤2)、种子二级培养：按照4-6％接种比加入步骤1)得到的种子液，在36-40℃下搅拌培养16-20h，搅拌转速90-110rpm，通过部件泵流加2mol/L的NaOH来控制为7.3，通入氮气维持种子罐压力在0.05MPa；
步骤3)、发酵罐培养：将二级种子罐培养得到的所述一级种子液全部泵入发酵罐，35-40℃下搅拌培养15-20h，搅拌转速180-240rpm，pH采用2mol/L的NaOH通过种子罐补碱泵自动控制在4.4-4.6，且通入氮气维持发酵罐压力在0.05MPa；培养期间通过补料泵确保罐内葡萄糖浓度在2g/L以上；
步骤4)、菌体分离、复配：通过碟片式离心机分离菌体，之后用无菌水洗涤2次得到菌泥，按照质量比5-10:1的比例将菌泥与菌泥保护剂混合乳化，进行冷冻干燥。
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